1. Transfect cells with plasmids encoding heat stable secreted
alkaline phosphatase (AP) gene as reporter.
2. 24-48 hours after transfection, harvest 200 ml of medium.
3. Heat the medium to 65 °C for 30 minutes to inactivate any endogenous phosphatases.
4. Spin down cell debris.
5. Transfer 50 ml to 96-well plate wells.
6. Add 150 µl of substrate (5 µM p-nitrophenyl phosphate (PNPP) in 50 mM Tris-HCl, pH 8.2) to each well.
Adding 25 µl 0.05% Zwittergent (in PBS) may reduce background.
7. If quantitative measurment is required, use an AP standard to generate a standard curve.
8. Incubate at room temperature or 37 °C for 30 minutes or more.
9. Read the plate at 405 nm.